CN103436625A

CN103436625A - 6 deafness susceptibility gene locus typing/mutation proportion detection kit

Info

Publication number: CN103436625A
Application number: CN2013104107817A
Authority: CN
Inventors: 步迅; 夏子芳; 刘艳艳; 张全芳
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-09-11
Filing date: 2013-09-11
Publication date: 2013-12-11
Anticipated expiration: 2033-09-11
Also published as: CN103436625B

Abstract

The invention discloses a 6 deafness susceptibility gene locus typing/mutation proportion detection kit. The kit comprises an amplification reagent and a series of standard samples, wherein the amplification reagent comprises a reaction mixture of a PCR (Polymerase Chain Reaction) buffer solution, MgCl2 and dNTPs, a Taq enzyme, ultrapure water, high-specific amplification GJB2, SLC26A4 and a primer mixture of a mitochondrion 12SrDNA; and the series of standard samples include 235delC, 299delAT, 2168A>G and IVS7-2A>G typing standard samples, a 1494C>T mutation proportion standard sample and a 1555A>G mutation proportion standard sample. The aims of reporting the mutation proportions of 1494C>T and 1555A>G and realizing the 6-locus typing detection by using a four-channel/four-color dye are achieved for the first time by using a fluorescent quantitative PCR detection method.

Description

6 deaf sick susceptibility loci somatotypes/mutant proportion detection kit

Technical field

The present invention relates to 6 deaf sick susceptibility loci somatotypes/mutant proportion detection kit, belong to technical field of biological.

Background technology

The paathogenic factor research of the hearing and speech handicapped person being carried out in world wide shows, about 60-80% patient's the cause of disease is relevant with inherited genetic factors, and wherein the clinical study data of developed country show, hereditary hearing impairment accounts for 80% in deafness patient.Therefore in recent ten years, the pathogenesis of hereditary hearing impairment and the research of molecular epidemiology thereof become one of most important content of deaf sick research.Along with the Human Genome Project completes, the location of deaf ospc gene and clone have obtained huge progress, and the molecule genetics research of deaf disease and the data of molecular epidemiology make investigators progressively recognize that deaf sick susceptibility gene mutation safeguarding that hearing is healthy and finding the importance of hearing in abnormal.

In the deaf-related gene of having located and having cloned at present, GJB2 is modal tumor susceptibility gene, in congenital severe deafness patient, accounts for 30-50%.At present, at this gene, had been found that 100 various mutations types, in China, common mutant form is 235delC, and it accounts for 74.14% in all disease cause mutations, and approximately 22.2% Chinese non-syndromic hearing loss patient is relevant with this sudden change.The mutation allele frequency of GJB2299_300delAT is about 3% in addition.The large vestibular aqueduct syndrome that SLC26A4 gene and arc are vertical and Pendred syndrome (aqueductus vestibuli enlarges or companion's inner ear malformations, nerve deafness and thyrocele) in close relations, show as clinically congenital or acquired character is deaf, deaf occur or increase the weight of with wound, catch a cold relevant.In 95 routine single patient's aqueductus vestibulis enlarge the patient of family, 97.9% (93/95) the sudden change with SLC26A4 gene, in 38 kinds of mutation types finding, IVS7-2A > G, 2168A > G enlarges in patient SLC26A4 transgenation and occupies higher ratio at Chinese aqueductus vestibuli.The gene type result of these chromosome mutations has very important directive significance for seriousness and the guidance fertility heredity of analysing patient's condition.

The ototoxicity of medicine is an important factor that causes hearing loss before language, part and plastosome 12S rRNA gene 1555A > the G sudden change is relevant, and this sudden change can increase the susceptibility of cochlea to aminoglycoside drug.In the U.S., in the relevant hearing loss patient of ototoxic drug, 10% there is 12S rRNA gene 1555A the G sudden change, before U.S.'s language in the hearing loss patient, 1555A > G sudden change patient's sickness rate is about 1/20000-1/40000.In Spain, 12S rRNA gene 1555A > the G sudden change is relevant with the familial nonsyndromic hearing loss of 15-20%, the auditory dysesthesia that many old family members also can occur so suddenly change and cause even without the use aminoglycoside drug.And in China, the sickness rate of drug induced deafness has exceeded original imagination, in a series of article report, in the deafness patient that discovery is found in outpatient service, approximately 5% patient is due to 12S rRNA gene 1555A > G sudden change causes, and, in such specific group of school for deaf-mutes, up to 12% patient, be due to 12S rRNA gene 1555A G sudden change contact aminoglycoside drug causes deaf.In Chinese colony, also found 12S rRNA gene 1494C simultaneously > relation of T sudden change and drug induced deafness, at present at least had been found that three large familys are due to 1494C > T suddenlys change and causes.1555A > G sudden change and 1494C > often hearing is normal at birth for T sudden change person, is difficult to found in advance and predict by universal newborn hearing screening, but has the hidden danger that hearing loss occurs because of the use of ototoxic drug.Due to mitochondrial heterogeneous feature, the ratio difference of the mitochondrial mutations that different individualities shows.Large quantity research shows, 1555A > G and 1494C > there is certain dependency in the ratio height that occupies of T sudden change plastosome with the state of an illness, and the individuality that the plastosome that usually suddenlys change proportion is high shows severe deafness, and accounting is low shows slight deafness or normal.Even but the sudden change accounting is very low, still exists medicine to cause deaf danger.Therefore, need on the one hand the test kit that can tell mutant proportion for the relevant sudden change of the deaf disease of plastosome provides research means more easily, require on the other hand these reagent good Detection capability to be arranged to low mutant proportion sample.

Gene tester commonly used has at present: direct Sequencing (direct sequencing, DS), Ligase detection reaction (1igase detection reaction, LDR), restriction fragment length polymorphism analysis (restriction fragment length polymorphism, RFLP), dhplc analysis (denaturing high performance liquid chromotography, DHPLC), gene chip.All there is different defects in these methods, such as complex operation, result, are difficult for the problems such as interpretation, poor repeatability, false negative false positive be many.For the gene type of chromosome mutation, general fluorescence quantitative kit often needs same sample is carried out to the multitube detection, could obtain the somatotype information more than 2.Mutant proportion for Mitochondrial DNA detects, and mass spectroscopy can detect the sudden change of 5% left and right, and order-checking and gene chip can only reach 20% left and right for the mitochondrial selective power of sudden change, and indistinguishable goes out the sample of lower mutant proportion.

It is detected object that above-mentioned 6 deaf sick susceptibility locis are take in the present invention, amplification and fluorescent quantitation by above-mentioned deaf susceptibility loci detect, contrast with gene type/mutant proportion standard substance, can filter out the individuality that contains above-mentioned site mutation, determine ratio and the genotype of various sudden changes simultaneously.To the detection of deaf sick tumor susceptibility gene, especially significant to the examination of newborn infant's deaf gene; Utilize first the method for fluorescence quantitative PCR detection to realize 1494C > T, 1555A > the mutant proportion report of G and the target that four-way/tetra-look dyestuffs are realized 6 site somatotypes detections, greatly saved manpower and materials and time.The highly sensitive high specific detects simultaneously, provides the somatotype information of the relevant sudden change of karyomit(e), for plastosome 1555A > G and 1494C > selective power of T sudden change reached 0.1%.Stopped pipe detects and to have prevented the pollution of uncapping and detecting operation and producing, for clinical diagnosis and association area scientific research provide reliable method.

Summary of the invention

The object of the invention is to provide a kind of 6 hot spot mutations that can simultaneously detect in 1 hour in modal 3 deaf-related genes of Chinese, reports ratio and the genotypic fluorescence detection reagent kit of various sudden changes simultaneously.

To achieve these goals, the technical scheme of taking: a kind of 6 deaf sick susceptibility loci somatotypes/mutant proportion detection kit, this test kit comprises amplifing reagent and series of standards product;

Described amplifing reagent comprises: PCR damping fluid, MgCl ₂reaction mixture with dNTPs, the Taq enzyme, ultrapure water, high specific amplification GJB2:235delC, 299delAT, SLC26A42168A>G, IVS7-2A>G, plastosome 12SrDNA:1494C>T, 1555A>primer mixture of G, carry out the probe mixture of specific detection, the general probe mixture that the human gene group DNA is detected to above-mentioned site;

Described series of standards product comprise: 235delC somatotype standard substance, 299delAT somatotype standard substance, 2168A>G somatotype standard substance, IVS7 _-2A>G somatotype standard substance, 1494C>T mutant proportion standard substance, 1555A>G mutant proportion standard substance.

Described for to GJB2:235delC, 299delAT, SLC26A4:2168A > G, IVS7-2A > G, plastosome 12SrDNA:1494C > T, 1555A > G sudden change carries out the probe of specific detection, the general probe that the human gene group DNA is detected is divided into four groups, adopt respectively four kinds of different luminophores to carry out mark to 5 ' end of each group probe, the quenching group of 3 ' end can be identical or different fluorescence dye.

Four groups of being divided into of described probe are: be one group for the probe that specific detection is carried out in sudden change to GJB2:235delC, 299delAT, adopt same group of luminous-quenching group; SLC26A4:2168A > G, IVS7-2A > the G sudden change probe that carries out specific detection is one group, adopts same group of luminous-quenching group; Plastosome 12SrDNA:1494C > T, 1555A > the G sudden change probe that carries out specific detection is one group, adopts same group of luminous-quenching group; The general probe that the human gene group DNA is detected is one group, adopts same group of luminous-quenching group.

Described 5 ' the fluorescence dye of holding different luminophores to use can be: ALEXA Fluor350, FAM, TET, HEX/JOE/VIC, Cy3, TAMRA, ROX ,/Texas Red, Cy5; 3 ' the fluorescence dye of holding identical or different quenching group to use can be: BHQ1, BHQ2, TAMRA, DABCYL.

Use described test kit to carry out the condition of PCR composite amplification reaction: the pH value of pcr amplification system is 8.0-9.0, magnesium ion concentration is 1.5-3.5mM, the final concentration of 4 kinds of dNTP is respectively 200-300 μ M, the consumption of Taq enzyme is 0.1-0.4U/ μ L, and the primer in the primer probe mixture, the final concentration of probe are 0.2-0.4 μ M.

While using described test kit to carry out pcr amplification, amplification elementary reaction in a composite amplification reaction system, increase GJB2:235delC, 299delAT simultaneously; SLC26A4:2168A > G, IVS7-2A > G; 12SrDNA:1494C > T, 1555A > G.

For GJB2:235delC, 299delAT, SLC26A4:2168A > G, IVS7-2A > G, plastosome 12SrDNA:1494C > T, 1555A > primer that detects of G is:

SEQ?NO.1：5’-GTAAGTTGGGTGCTTTGTGTTAAG-3’

SEQ?NO.2：5’-GCCCTGAAGCGCGTACA-3’；

SEQ?NO.3：5’-TGGGATGGATTTAACAATGCC-3’

SEQ?NO.4：5’-GTTAGAAAGTTCAGCATTATTTGGTTG-3’；

SEQ?NO.5：5’-AATGGAACCTTGACCCTCTTGA-3’

SEQ?NO.6：5’-TGTGAIAGAAAAGCTGGAGCAATG-3’；

SEQ?NO.7：5’-ACGTGTGCTACGATCACTACTTCC-3’

SEQ?NO.8：5’-CTCCTCGATGTCCTTAAATTCACTC-3’；

For plastosome 12SrDNA1555A > probe that detects of G sudden change is:

SEQ?NO.9：5’-AGTACACTTACCATGTTACGACTTGcCTCCTC-3’

For plastosome 12SrDNA1494C > probe that detects of T sudden change is:

SEQ?NO.10：5’-GTGAAGTATACTTGAGGAGaGTGACGGGAC-3’；

For GJB2:235delC, 299delAT, SLC26A4:2168A > G, IVS7-2A > probe that detects of G sudden change is:

SEQ?NO.11：5’-AAATGGCAGTAGCAATTATCGTCcGAA-3’

SEQ?NO.12：5’-CTTGGTTCTGTAGAIAGAGTATAGCATCAcGGAC-3’；

SEQ?NO.13：5’-ACATCCGGCTATGGGCCTGC-3’

SEQ?NO.14：5’-CCTTGATGAACTTCCTCTTCTTCTCGTCTC-3’；

For the general probe that the human gene group DNA is detected, be:

SEQ?NO.15：5’-GAATGTGTCCTTTCTAATGTTGTCGTC-3’；

The composite amplification in described each site adopts polymerase chain reaction to realize, adopts quantitative fluorescent PCR to detect in real time, by the synchronous amplification with standard substance and analysis, draws institute's mark somatotype information or mutant proportion information originally.

The human gene group DNA that wherein detected is: use Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to process to the source sample DNA obtained; Described source sample is: derive from the mankind: filter paper blood cake/buccal swab sample, FTA card blood cake/buccal swab sample, buccal swab sample, blood/trace, tissue, amniotic fluid.

While using described test kit to carry out pcr amplification, the amplification elementary reaction carries out on the PCR of any model instrument, amplification program: 94-98 ℃ 1-5min; The 94-98 of 45 circulations ℃ 5-10s, 55-65 ℃ of 30-50s.

Use with fluorescent mark and mix the primer of modification through the LNA nucleoside monomers, improve the Tm value of probe, shortened probe length, strengthened the insight of probe to point mutation, thereby increased sensitivity and the specificity of probe, prevented false positive and false-negative appearance.

Under equal DNA concentration background, when guaranteeing the successful augmentation detection of each site mutation, designed and screened primer and the probe with different amplification efficiencies for each site, make the Ct value in each site different, by the contrast with standard substance amplification collection of illustrative plates, thereby judge, be which kind of site mutation.

The accompanying drawing explanation

Fig. 1 is 235delC somatotype standard substance, 299delAT somatotype standard substance, 2168A > G somatotype standard substance, IVS7-2A > G somatotype standard substance carry out fluorescent quantitation and collect the typical curve that data obtain;

Fig. 2 is that 1555 mutant proportion standard substance, 1494 mutant proportion standard substance carry out the typical curve that fluorescent quantitation collection data obtain;

Fig. 3 is that test kit of the present invention carrys out the detection collection of illustrative plates (not having the passage that signal rises not mark) of source DNA (0.5ng/20uL system) to the normal individual blood cake;

Fig. 4 is that test kit of the present invention carrys out the detection collection of illustrative plates (not having the passage that signal rises not mark) of source DNA (0.5ng/20uL system) to the individual blood cake of GJB2235delC sudden change;

Fig. 5 is that test kit of the present invention carrys out the detection collection of illustrative plates (not having the passage that signal rises not mark) of source DNA (1ng/20uL system) to GJB2:299delAT mutated individual blood cake;

Fig. 6 is that test kit of the present invention carrys out the detection collection of illustrative plates (not having the passage that signal rises not mark) of source DNA (5ng/20uL system) to the individual blood cake of 2168 sudden changes;

Fig. 7 is that test kit of the present invention carrys out the detection collection of illustrative plates (not having the passage that signal rises not mark) of source DNA (5ng/20uL system) to IVS7-2 mutated individual blood cake;

Fig. 8 is that test kit of the present invention is to 12S rRNA1555A > the individual blood cake of G sudden change carrys out the detection collection of illustrative plates of source DNA (10ng/20uL system) (not having the passage that signal rises not mark);

Fig. 9 is that test kit of the present invention is to 12S rRNA1494C > T mutated individual blood cake carrys out the detection collection of illustrative plates (not having the passage that signal rises not mark) of source DNA (2.5ng/20uL system).

Embodiment

Below in conjunction with the drawings and specific embodiments, the invention will be further described, but not as a limitation of the invention.

Embodiment 1 test kit of the present invention detects the DNA sample of sudden change and normal individual

Probe 5 ' the end detected for deaf sick tumor susceptibility gene GJB2:235delC, 299delAT adopts the FAM fluorochrome label, for SLC26A4 mutantional hotspot: 2168A > G, IVS7-2A > the probe 5 ' end that detects of G sudden change adopts the HEX fluorochrome label, for 12S rRNA1494C > T, 1555A > the probe 5 ' end that detects of G sudden change adopts the ROX fluorochrome label, and the probe 5 ' end detected for the somatotype standard substance adopts the Cy5 fluorochrome label.

1, sample to be tested is 1000 parts, all uses the technological method of " DNA extraction-pcr amplification-order-checking " to carry out the order-checking detection to each site.Wherein, IVS7-2A > 10 parts, G sudden change sample, 10 parts, GJB2235delC sudden change sample, 12S rRNA1555A > 10 parts, G sudden change sample, 1494C > 1 part, T sudden change sample, 2168A > 1 part, G sudden change sample, 1 part, GJB2299delAT sudden change sample.

2, the extracting genome DNA of sample

Chelex extraction method: cut 1～3mm blood cake (sample comes from the XX hospital laboratory) and be placed in the 1.5mL centrifuge tube, add sdH ₂o1mL, vibrate centrifugal, abandons supernatant liquor, and repeating step twice, abandon supernatant liquor, and draw 200 μ L with the rifle head of cutting fast after the 5%Chelex-100 concussion is suspended and add in centrifuge tube, the vibration several seconds.After 56 ℃ of water bath heat preservation 30min, the vibration several seconds.95 ℃ of boiling water bath 10min, slightly vibrate the several seconds.The centrifugal 5min of 2000rpm, the DNA for extracting in supernatant.

3, the detection analysis of amplification and amplified production

(1) pcr amplification system:

(2) amplification program on ABI StepOne Plus type quantitative real time PCR Instrument: 95C3min; 95C5s, 60C35s (collection fluorescent signal), be 45cycles.

(3) detect and analyze

Get 235delC somatotype standard substance (235 heterozygous mutant type DNA under 10ng DNA background), 299delAT somatotype standard substance (299 heterozygous mutant type DNA under 10ng DNA background), 2168A > G somatotype standard substance (235 heterozygous mutant type DNA under 10ng DNA background), IVS7-2A > G somatotype standard substance (299 heterozygous mutant type DNA under 10ng DNA background) dilute respectively for 5ng/uL, 1ng/uL, 0.5ng/uL, getting respectively 1uL joins in amplification system as template, carry out fluorescent quantitation and collect data, be figure according to each concentration and corresponding Ct value, obtain the typical curve of Fig. 1.

1555 mutant proportion standard substance (1555 different mutant proportions under 10ng DNA background), 1494 mutant proportion standard substance (1494 different mutant proportions under 10ng DNA background) are got respectively to 1uL and joined in amplification system as template, carry out fluorescent quantitation and collect data, be figure according to each concentration and corresponding Ct value, obtain the typical curve of Fig. 2.

Fig. 3 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (0.5ng/20uL system) to the normal individual blood cake: obtain two passages of human gene group DNA's probe (Cy5) and signal and sigmoid curve detected, the Ct value is 35.11, shows that the concentration of this DNA sample is about 0.5ng/uL; 235delC probe, 299delAT probe in detecting passage (FAM), 2168A > G probe, IVS7-2A > G probe in detecting passage (HEX), and 1555A > G probe, 1494C > 3 sense channels of T probe in detecting passage (ROX) all do not have sigmoid curve, (not having the passage that signal rises not mark), show that this sample is the normal individual source that all there is not sudden change in above-mentioned 6 sites.

Fig. 4 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (0.5ng/20uL system) to the individual blood cake of GJB2:235delC sudden change: obtain two passages of human gene group DNA's probe (Cy5) and signal and sigmoid curve detected, the Ct value is 35.07, shows that the concentration of this DNA sample is about 0.5ng/uL; HEX, ROX passage do not have signal to rise (not marking on figure); The FAM passage detects signal and sigmoid curve, and the Ct value is 31.19, and contrast Fig. 1: the Ct value of 0.5ng human gene group DNA 299delAT heterozygous mutant type should be 37.09, so be not 299 site mutations; It should be 32.09 that contrast Fig. 1 finds the Ct value of 0.5ng human gene group DNA 235delC heterozygous mutant type, shows that this sample should be 235delC homozygous mutation type.

Fig. 5 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (1ng/20uL system) to GJB2:299delAT mutated individual blood cake: obtain two passages of human gene group DNA's probe (Cy5) and signal and sigmoid curve detected, the Ct value is 34.02, shows that the concentration of this DNA sample is about 1ng/uL; HEX, ROX passage do not have signal to rise (not marking on figure); The FAM passage detects signal and sigmoid curve, and the Ct value is 36.19, and contrast Fig. 1 determines that this sample should be 1ng/uL299delAT heterozygous mutant type.

Fig. 6 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (5ng/20uL system) to the individual blood cake of 2168 sudden changes: obtain human gene group DNA's probe (Cy5) passage and signal and sigmoid curve detected, the Ct value is 31.82, shows that the concentration of this DNA sample is about 5ng/uL; FAM, ROX passage do not have signal to rise (not marking on figure); The HEX passage detects signal and sigmoid curve, and the Ct value is 24.01, and contrast Fig. 1 determines that this sample should be 5ng/uL2168A > G heterozygous mutant type.

Fig. 7 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (5ng/20uL system) to IVS7-2 mutated individual blood cake: obtain human gene group DNA's probe (Cy5) passage and signal and sigmoid curve detected, the Ct value is 31.85, shows that the concentration of this DNA sample is about 5ng/uL; FAM, ROX passage do not have signal to rise (not marking on figure); The HEX passage detects signal and sigmoid curve, and the Ct value is 19.99, and contrast Fig. 1 determines that this sample should be 5ng/uL IVS7-2A > G homozygous mutation type.

Fig. 8 is that test kit of the present invention is to 12S rRNA1555A > the individual blood cake of G sudden change carrys out the detection collection of illustrative plates of source DNA (10ng/20uL system): obtains two passages of human gene group DNA's probe (Cy5) and signal and sigmoid curve detected, the Ct value is 30.81, shows that the DNA background concentration of this sample is about 10ng/uL; FAM, HEX passage do not have signal to rise (not marking on figure); The ROX passage detects signal and sigmoid curve, and the Ct value is 23.21, and contrast Fig. 2 determines that this sample should be 1555A > the G mutant proportion is 100%.

Fig. 9 is that test kit of the present invention is to 12S rRNA1494C > T mutated individual blood cake carrys out the detection collection of illustrative plates of source DNA (2.5ng/20uL system): obtains two passages of human gene group DNA's probe (Cy5) and signal and sigmoid curve detected, the Ct value is 32.87, shows that the concentration of this DNA sample is about 2.5ng/uL; FAM, HEX passage do not have signal to rise (not marking on figure); The ROX passage detects signal and sigmoid curve, and the Ct value is 22.89, and contrast Fig. 3 (mutant proportion that under the 10ng background, Ct value is 23 left and right correspondences is 10%), show the 1494C of this sample > the T content that suddenlys change is about 40%.

With sequence measurement, compare: the homologous genes seat somatotype result to the sample in same source is consistent.

10 * PCR damping fluid of different pH used in above embodiment, with the Tris-HCl damping fluid preparation of different pH values, the Tris-HCl concentration in 1 * PCR damping fluid is 10mM, KCl concentration is 50mM; In the present invention, Taq polysaccharase used and other reagent and material are commercially available prod.

Claims

1.6 deaf sick susceptibility loci somatotype/mutant proportion detection kit, is characterized in that comprising amplifing reagent and series of standards product; Described amplifing reagent comprises: PCR damping fluid, MgCl ₂reaction mixture with dNTPs, the Taq enzyme, ultrapure water, high specific amplification GJB2:235delC, 299delAT, SLC26A4:2168A>G, IVS7-2A>G, plastosome 12SrDNA:1494C>T, 1555A>primer mixture of G, carry out the probe mixture of specific detection, the general probe mixture that the human gene group DNA is detected to above-mentioned site; Described series of standards product comprise: 235delC somatotype standard substance, 299delAT somatotype standard substance, 2168A>G somatotype standard substance, IVS7-2A>G somatotype standard substance, 1494C>T mutant proportion standard substance, 1555A>G mutant proportion standard substance.

2. test kit according to claim 1 is characterized in that:

SEQ?NO.1：5’-GTAAGTTGGGTGCTTTGTGTTAAG-3’

SEQ?NO.2：5’-GCCCTGAAGCGCGTACA-3’；

SEQ?NO.3：5’-TGGGATGGATTTAACAATGCC-3’

SEQ?NO.4：5’-GTTAGAAAGTTCAGCATTATTTGGTTG-3’；

SEQ?NO.5：5’-AATGGAACCTTGACCCTCTTGA-3’

SEQ?NO.6：5’-TGTGAIAGAAAAGCTGGAGCAATG-3’；

SEQ?NO.7：5’-ACGTGTGCTACGATCACTACTTCC-3’

SEQ?NO.8：5’-CTCCTCGATGTCCTTAAATTCACTC-3’；

For plastosome 12SrDNA1555A > probe that detects of G sudden change is:

SEQ?NO.9：5’-AGTACACTTACCATGTTACGACTTGCCTCCTC-3’

For plastosome 12SrDNA1494C > probe that detects of T sudden change is:

SEQ?NO.10：5’-GTGAAGTAIACTTGAGGAGaGTGACGGGAC-3’；

SEQ?NO.11：5’-AAATGGCAGTAGCAATTATCGTCCGAA-3’

SEQ?NO.12：5’-CTTGGTTCTGTAGAIAGAGTATAGCATCACGGAC-3’；

SEQ?NO.13：5’-ACATCCGGCTATGGGCCTGC-3’

SEQ?NO.14：5’-CCTTGATGAACTTCCTCTTCTTCTCGTCTC-3’；

For the general probe that the human gene group DNA is detected, be:

SEQ?NO.15：5’-GAATGTGTCCTTTCTAATGTTGTCGTC-3’；

Wherein the LNA nucleosides means with boldface type.

3. test kit according to claim 1 and 2, is characterized in that: described for GJB2:235delC, 299delAT, SLC26A4; 2168A > G, IVS7-2A > G, plastosome 12SrDNA:1494C > T, 1555A > probe that G sudden change detects, 5 ' end and the 3 ' end of every probe all carry out fluorochrome label, and 5 ' end is quenching group for luminophore and 3 ' end.

4. test kit according to claim 1 and 2, it is characterized in that: described for to GJB2:235delC, 299delAT, SLC26A4:2168A > G, IVS7-2A > G, plastosome 12SrDNA:1494C > T, 1555A > G sudden change carries out the probe of specific detection, the general probe that the human gene group DNA is detected is divided into four groups, adopt respectively four kinds of different luminophores to carry out mark to 5 ' end of each group probe, the quenching group of 3 ' end can be identical or different fluorescence dye.

5. require described test kit according to right 4, it is characterized in that: four groups of being divided into of probe are: be one group for the probe that specific detection is carried out in sudden change to GJB2:235delC, 299delAT, adopt same group of luminous-quenching group; SLC26A4:2168A > G, IVS7-2A > the G sudden change probe that carries out specific detection is one group, adopts same group of luminous-quenching group; Plastosome 12SrDNA:1494C > T, 1555A > the G sudden change probe that carries out specific detection is one group, adopts same group of luminous-quenching group; The general probe that the human gene group DNA is detected is one group, adopts same group of luminous-quenching group.

6. require described test kit according to right 4, it is characterized in that: the 5 ' fluorescence dye of holding different luminophores to use can be: ALEXA Fluor350, FAM, TET, HEX/JOE/VIC, Cy3, TAMRA, ROX ,/Texas Red, Cy5; 3 ' the fluorescence dye of holding identical or different quenching group to use can be: BHQ1, BHQ2, TAMRA, DABCYL.

7. according to right 1, require described test kit, it is characterized in that: comprise the series of standards product: 235delC somatotype standard substance, 299delAT somatotype standard substance, 2168A G somatotype standard substance, IVS7-2A G somatotype standard substance, 1494C T mutant proportion standard substance, 1555A G mutant proportion standard substance.

8. require described test kit according to right 1 or 7, it is characterized in that: the somatotype standard substance are: 235delC heterozygous mutation DNA sample 1 pipe of 10ng/uL, homozygous mutant DNA sample 1 pipe of the 235delC of 10ng/uL, 299delAT heterozygous mutation DNA sample 1 pipe of 10ng/uL, homozygous mutant DNA sample 1 pipe of the 299delAT of 10ng/uL, the 2168A of 10ng/uL > IVS7-2A of G heterozygous mutation DNA sample 1 pipe, 10ng/uL > homozygous mutant DNA sample 1 pipe of G.

9. require described test kit according to right 1 or 7, it is characterized in that: 1494C T mutant proportion standard substance are: the people DNA sample of 7 pipe 10ng/uL, the 1494C of each pipe > the T mutant proportion is 100%, 50%, 20%, 10%, 5%, 1%, 0.5%, its preparation method is for by 10ng/uL100%1494C > the DNA sample of T sudden change mixes by a certain percentage 10ng/uL and do not contain 1494C > in the DNA sample of T sudden change.

10. require described test kit according to right 1 or 7, it is characterized in that: 1555A G mutant proportion standard substance are: the people DNA sample of 7 pipe 10ng/uL, the 1555A of each pipe > the G mutant proportion is 100%, 50%, 20%, 10%, 5%, 1%, 0.5%, its preparation method is for by 10ng/uL100%1555A > the DNA sample of G sudden change mixes by a certain percentage 10ng/uL and do not contain 1555A > in the DNA sample of G sudden change.

11. the described test kit of claim 1 is applied to the detection of deaf sick tumor susceptibility gene, it is characterized in that by the deaf sick tumor susceptibility gene GJB2:235delC of quantitative fluorescent PCR specific detection, 299delAT; SLC26A4:2168A > G, IVS7-2A > G; 12SrDNA:1494C > T, 1555A > G; The primer probe detected for GJB2235delC is: SEQ NO.7, SEQ NO.8, SEQ NO.13; The primer probe detected for GJB2299delAT is: SEQ NO.7, SEQ NO.8, SEQ NO.14; For 12S rRNA1555A > the primer probe that detects of G sudden change is: SEQ NO.1, SEQ NO.2, SEQ NO.9; For 12S rRNA1494C > the primer probe that detects of T sudden change is: SEQ NO.1, SEQ NO.2, SEQ NO.10; For IVS7-2A > the primer probe that detects of G is: SEQ NO.3, SEQ NO.4, SEQ NO.11; For SLC26A4.2168A > the primer probe that detects of G sudden change is: SEQ NO.5, SEQ NO.6, SEQ NO.12; For the primer probe that the human gene group DNA is detected, be: SEQ NO.5, SEQ NO.6, SEQ NO.15.The composite amplification in described each site adopts polymerase chain reaction to realize, adopts quantitative fluorescent PCR to detect in real time, by the synchronous amplification with standard substance and analysis, draws institute's mark somatotype information or mutant proportion information originally.